Systems allowing inducible control of protein activities, allowing precise manipulation of signaling pathways, protein expression and other processes, have transformed experimental biology. While a number of studies have used chemicals to perturb such processes, in recent years scientists have developed light-responsive 'optogenetic' tools, which allow precise spatio- temporal control of activity. In recent work, we have developed a light-induced dimerization platform based on cryptochrome (CRY2) and its interacting partner CIB1 that allows control of protein-protein interactions and localization with light. Our initial characterization indicates that this system has distinct properties that will facilitate use in model organisms and in vivo, as it has fast kinetics, is reversible, and is entirly genetically encoded. The broad aims of this project are to further develop the CRY2-CIB1 dimerization technology such that it can be used in a broad range of biological applications. Specifically, we aim to engineer improved dimerizers that are smaller, monomeric, more tightly controlled, and have improved dynamic range (Aim 1). In Aim 2, we will generate a 'toolkit' of optimized light-activated proteins, including a recombinase, a transcriptional activator, and a protease. Together, these enzymes will enable diverse approaches for regulation of biological activity at the levels of gene, RNA, and protein. A robust light-activated Cre recombinase, for example, would have enormous significance to neuroscience, developmental biology, and other fields, and would have widespread use in vivo in a variety of model systems. In Aim 3, we will examine the properties of the CRY2-CIB1 system and light-activated enzymes for control of biological systems. PUBLIC HEALTH RELEVANCE: The goal of this research is to develop novel tools for light-inducible control of protein activities. These tools, allowing precise spatial, temporal, and dose dependent control of biological processes, will open up vast new experimental avenues, allowing better understanding of pathways important in health and disease.